Bacillus thuringiensis is a spore-forming Gram-positive bacterium. During sporulation, B. thuringiensis produces proteinaceous inclusions which are composed of proteins known as insecticidal crystal proteins (ICPs), Cry proteins, or delta-endotoxins. These proteins are toxic to a variety of insect species including orders Lepidoptera, Coleoptera, Diptera, Hemoptera, Hymenoptera, Orthoptera, and Mallophaga and to nematodes, mites, and protozoa (Beegle and Yamamoto, Can. Entomol. 124:587-616; Feitelson, Advanced Engineered Pesticides (L. Kim, ed.), Marcel Dekker, Inc., New York (1993), pp. 63-71; Feitelson, et al., Bio/Technology 10:271-275; U.S. Pat. No. 4,948,734 (1990)). Due to their high specificity for particular insect pests and their safety for man and the environment, ICPs have been used as biopesticides for the last three decades. Using molecular genetic techniques, numerous delta-endotoxin genes have been isolated and their DNA sequences determined. The cloning and sequencing of a number of 6-endotoxin genes from a variety of B. thuringiensis strains has been described and are summarized by Schnepf et al. (Microbiol. Mol. Biol. Rev. 62:775-806, Bacillus thuringiensis And Its Pesticidal Crystal Proteins, 1998). The nomenclature and appearance of newly identified genes is summarized and regularly updated at by Crickmore, N., Zeigler, D. R., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J, Bravo, A. and Dean, D. H. “Bacillus thuringiensis toxin nomenclature” at the University of Sussex Department of Biology web site biols.susx.ac.uk/home/neil_crickmore/bt. These genes have been used to develop certain genetically engineered B. thuringiensis products that are in commercial use. Recent developments have seen new δ-endotoxin delivery systems developed, including genetically engineered plants that contain and express δ-endotoxin genes. Bacillus thuringiensis is a key source of genes, which when modified can be used for transgenic expression to provide pest resistance in plants.
B. thuringiensis strains are classified into subspecies or varieties, based on biochemical and serological criteria (de Barjac, Entomophaga 7: 5-61 (1962); de Barjac, Proceedings of the IIIrd International Colloquium on Invertebrate Pathology (C. C. Payne and H. D. Burges, eds.), Society for Insect Pathology, U.K., 451-453 (1982)) Each subspecies may produce one or several insecticidal protein toxins. To date, approximately 172 δ-endotoxins belonging to 28 classes have been identified. There is also a nonprotein toxin, the β-exotoxin, secreted by some B. thuringiensis strains. This toxin, which is assayed on house fly larvae (Sêbesta et al., “Thuringiensin, the β-exotoxin of Bacillus thuringiensis,” in W.H. Burgess (ed.), Microbial Control of Pests and Plant Diseases, 1970-1980, Academic Press, Inc., New York, pp. 249-281 (1981)), is not as selective as the δ-endotoxins.
Extensive studies have been carried out with B. thuringiensis subspecies that produce proteinaceous inclusions during sporulation. The inclusions are often bipyramidal, but some are cuboidal or multifaceted, and there is a wide variety of other morphologies. Some strains contain more than one type of inclusion in each cell. These inclusions are present within the mother cell adjacent to the spore, but in a few subspecies, they are localized within the exosporium (Aronson et al., Bacteriol. Rev. 40:360-402 (1976)). Inclusions are released, as is the spore, upon cell lysis.
Bacillus strains can have a chromosomal genome size of 2.4 to 5.7 Mbp (Carlson, et al., Appl. Environ. Microbiol. 60: 1719-1725 (1994)). Physical maps of chromosomes of two B. thuringiensis strains, B. thuringiensis subsp. Berliner 1715 and B. thuringiensis subsp. Thuringiensis HD2, have been constricted and are estimated to be between 5.4 and 5.7 Mbp (Carlson, et al, Microbiol. 142: 1625-1634 (1996); Carlson and Kolstø, J. Bacteriol. 175: 1053-1060 (1993)). The total genomes of each of these two strains consist of one or more chromosomes, and a more variable component comprised of extrachromosomal elements (Carlson and Kolstø, Mol. Microbiol. 13:161-169 (1994)).
Most B. thuringiensis isolates have several extrachromosomal elements, some of them circular plasmids and others linear (Carlson, et al., Microbiol. 60: 1719-1725 (1994)). In general, crystal-protein genes are localized on large plasmids (ca. 40 to 200 Mda) of B. thuringiensis (Gonzalez, et al., Plasmid 5: 351-365 (1981); Carlton and Gonzalez, Molecular Biology of Microbial Differentiation, American Society for Microbiology, Washington, D.C. 246-252 (1985), Kronstad, et al., J Bacteriol. 154: 419-428 (1983)), and in some cases, more than one gene is present on a given plasmid (Aronson et al., Bacteriol. Rev. 40:360-402 (1976); Carlton et al., “The genetics and molecular biology of Bacillus thuringiensis,” in D. A. Dubnau (ed.), The Molecular Biology of the Bacilli, Vol. II, Academic Press, Inc., New York, pp. 211-249 (1985)). However, chromosomal crystal-protein genes have been reported in some B. thuringiensis strains (Carlson and Kolstø, J. Bacteriol. 175: 1053-1060 (1993), Klier, et al., EMBO J 1: 791-799 (1982), Kronstad, et al., J Bacteriol. 154: 419-428 (1983)).
Bacillus thuringiensis strains often contain multiple epigenetic elements which are known to harbor genes expressing vegetative insecticidal proteins (VIP's) and Bt crystalline insecticidal and nematocidal proteins. It is believed that many other Bt insecticidal/nematocidal genes are present within these sequences, some of which may only be expressed under conditions which cannot be artificially simulated, some of which may be cryptic, and some of which may be actively expressed but which have not been previously identified due to their limited availability as a result of very low levels of expression. Identification of whole or substantial portions of DNA sequences of individual plasmids would greatly facilitate identification of genes encoding novel insect inhibitory proteins. However, when one tries to isolate and purify plasmid DNA of a B. thuringiensis species for constructing genomic DNA libraries used in sequencing, it would be difficult to eliminate the contamination of chromosomal DNA. Such contamination would complicate greatly the sequencing effort of individual plasmids and subsequently hinder construction of genetic maps of individual plasmids of the B. thuringiensis species. Thus, it would be desirable to generate the complete DNA sequence of the chromosomal genome exclusive of epigenetic sequences of a B. thuringiensis species, because the complete DNA sequence of the chromosome could be used as a background to significantly minimize the interference of chromosomal DNA sequences in identification of whole or a substantial portion of individual plasmids and of novel genes encoding insect inhibitory proteins.
Furthermore, although it is unexpected that the complete DNA sequence of the Bacillus thuringiensis chromosomal genome exclusive of epigenetic sequences would provide a substantial number of Bt crystalline insecticidal/nematocidal and VIP genes for second generation insect/pest control in crop species, comparison of the open reading frames present within the Bacillus thuringiensis chromosomal genome with other bacterial genome sequences, in particular other. Bacillus species genomic sequences would allow the subtraction of common sequences and thus the identification of sequences novel and unique to Bacillus thuringiensis, and which may play a role in the regulation of expression or activity of genes encoding insecticidal proteins, and may also provide a plethora of useful genes for future insect resistance management technologies and applications. Therefore, it is advantageous to generate the complete DNA sequence of the chromosomal genome exclusive of epigenetic sequences of a B. thuringiensis species.
Chromosomal genome sequence information from B. thuringiensis allows comparisons of those sequences with sequences from other B. thuringiensis strains as well as comparisons with DNA sequences from other organisms, including plants, mammals such as humans, bacteria, and fungi such as yeasts. In addition, genome sequencing and mapping provides increased opportunities for identification and isolation of agents of commercial interest, as well as insight into mechanisms of genome interactions.